Microbial contamination of surfaces is a significant cause of morbidity and mortality. Rapid and routine procedures for quantitative determination of bacteria present on surfaces is frequently of vital importance, particularly in food processing and in hospitals. Food poisoning is often a result of microbial contamination of meat or food that occurs during processing. Contamination can be spread through contact of food with surfaces. In addition, spread of disease in hospitals and other facilities often occurs as a result of passage of infectious microbes on the surface of clothes or equipment.
Key feature of these applications is the requirement for rapid testing within minutes, a method that will overcome the potential contaminants from a variety of surfaces, a requirement for no cross-over in the results from one test to a second, and a need for both general and specific testing for microbes, that is, the ability to test for contamination by both total microbial counts and the ability to test for the presence of specific microbes.
Various methods have been utilized to measure microbial contamination on surfaces. Traditional procedures for assaying bacteria on surfaces are based on swabbing the surface followed by a culture of the swab for 24 to 48 hours in or on media that supports the growth of microbial species. The cultures are observed manually or with automated instrumentation to determine the number of colonies that have formed as an indicator of the number of microbes initially present on the surface. The disadvantages of this methodology are long assay times, requirements for specially trained personnel, and possible inadequate identification of the presence of certain potentially pathogenic microbes whose growth is not supported by the specific media or environment. In particular, it may be difficult to detect fungal contamination by this method. In addition, in many of the potential applications, the method does not give results in the time frame required for effective response.
Luminescent reactions have been utilized in various forms to detect bacteria in fluids and in processed materials. In particular, bioluminescent reactions based on the reaction of adenosine triphosphate (ATP) with luciferin in the presence of the enzyme luciferase to produce light, the "firefly" reaction have been utilized. Since ATP is present in all living cells including all microbial cells, this method can be used in a rapid assay to obtain a quantitative estimate of the number of living cells in a sample. Early discourses on the nature of the reaction, the history of its discovery, and its general area of applicability are provided by E. N. Harvey (1957), A History of Luminescence: From the Earliest Times Until 1900, Amer. Phil. Soc., Philadelphia Pa. and W. D. McElroy and B. L. Strehler (1949) Arch. Biochem.Biophys. 22:420-433. Alternatively, chemiluminescent detection by isoluminol or similar compounds has been used. This method is based on the detection of iron-containing substances in microbes.
Test procedures exemplifying the use of bioluminescent reactions for bacterial determinations and, specialized instrumentation for measurement of the associated light emission, are known and have been disclosed. Plakas (U.S. Pat. Nos. 4,013,418, 4,144,134, and 4,283,490) teaches a bioluminescent assay for the detection of bacteria in a sample including the steps of lysing non-bacterial cells, effecting filtration by positive pressure, washing, lysing bacterial cells and detecting ATP released with a luciferin/luciferase/Mg2+ reagent. This art in this patent does not deal with the specific problems associated with collection of material from a surface or with the detection of specific bacteria. No issue of the timing is mentioned and the invention as disclosed would require significant time.
Chappelle in U.S. Pat. No. 4,385,113 discloses a method for detection of bacteria in water based on bioluminescence. This test requires several hours to perform and is specifically addressed to the detection of total bacterial content in water.
Miller (PCT application US88/00852) discusses a similar assay for use with urine samples, but does not discuss the issues of collection from a surface and the assay timing is not specifically set forward in this application. Further, no method for detection of specific bacteria is elucidated.
Clendenning in his U.S. Pat. No. 3,933,592 discusses a method for bioluminescent detection of microbial contamination and in the examples refers to performing the procedure in less than 2 minutes. The procedure does not involve pre-treatment phases and the removal of somatic cell ATP.
AEgidius (U.S. Pat. No. 5,258,285) discloses a method for detection of bacterial concentration in a sample that utilizes a filtration step, a washing step to remove extraneous material including somatic cell ATP, establishing an extraction chamber in which the bacterial ATP is extracted, then transferring the material to a measuring chamber in which luciferin/luciferase/Mg2+ is added and the reaction measured. This method does not mention time. In addition, it utilizes separate chambers for washing, extracting the bacterial ATP, and measuring the reaction. This may potentially result in decreased sensitivity due to loss of the material in the process of transferring the solution from chamber to chamber. Further, the method does not describe a means of collecting a sample from a surface.
Detection of bacteria on surfaces poses additional issues not addressed in these previous methods. First and foremost is the method for collecting a sample to be compatible with these test devices and materials. The method must effectively retrieve the bacteria from the surface and result in a liquid suspension of the microbes.
A second issue of main concern is that surfaces being monitored often are contaminated with materials that may interfere with the detection of the microbes. One main interfering material that can be present on surfaces is somatic cells either from the food itself and including both animal and plant cells, or from the hands of an individual in contact with the surface. Since all living organisms including somatic cells contain ATP, the presence of these cells can mask or alter the reading obtained.
An additional source of interfering substances are those that interfere with the light producing reaction itself. These substances include a wide range of chemicals such as chlorine, oxidizing agents, free ATP, heavy metals, and other chemicals. As some of these chemicals are used for disinfecting of a surface, it is obvious that a reliable method for analyzing microbial contamination must include a means of eliminating these substances from the sample.
It is a further requirement in many cases in the food processing and hospital applications that the method for monitoring for microbial contamination of surfaces be rapid. For example, in the processing of beef carcasses, the carcasses are processed on a line and any testing of the material for microbial contamination must be performed within the time frame required for the carcass to move to further processing.
Previously disclosed luminescence based methodologies for microbial detection have not included any means for processing a sample from a surface and making a liquid suspension for testing. Further, the processes have required multiple devices or chambers for containment, filtration, and measurement of the reaction. Finally, the processes have not incorporated a disposable device that allows for minimizing cross-contaminations. Finally, in those assays for detecting specifically microbial ATP and other specific surface contaminants, previously disclosed inventions have relatively long time frames which are not consistent with on-line processing, quality control, and immediate verification of results.